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Developmental Competence, after Transfer to Recipients, of Porcine Oocytes Matured, Fertilized, and Cultured In Vitro

^a National Institute of Agrobiological Resources, Department of Genetic Resources II, Tsukuba, Ibaraki 305-8602, Japan ^b YS New Technology Institute, Inc., Ishibashi, Shimotsuga, Tochigi 329-0501, Japan ^c Azabu University, School of Veterinary Medicine, Sagamihara, Kanagawa 229-8501, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The objective of this study was to evaluate the developmental ability of early porcine embryos produced in vitro and transferred to recipient gilts. Porcine cumulus-oocyte complexes were matured in modified North Carolina State University-37 solution for 44–46 h (in vitro maturation, IVM). In vitro fertilization (IVF) was performed with frozen-thawed epididymal spermatozoa. Inseminated oocytes were cultured in vitro (IVC) for 0, 24, or 48 h in modified NCSU-37 solution. Embryos were surgically transferred to the oviducts of recipients in which estrus had been synchronized with eCG and hCG. On the 29th day post-IVF, the uteri of some recipients were surgically examined for pregnancy; then pregnant females were hysterectomized in order to examine number and weight of the fetuses. Developmental rates to fetuses for IVM/IVF oocytes cultured for 24 and 48 h were significantly lower (p < 0.05, 1.7% and 2.0%, respectively) than that of IVM/IVF oocytes without IVC (6.7%). However, the weights of fetuses (1.0–1.2 g) did not differ among the experimental groups. The other recipients were examined for pregnancy using an ultrasound pregnancy detector, and pregnant females were allowed to go to term. Healthy piglets were delivered by some recipients to which embryos cultured for 0 or 24 h had been transferred; however, no farrow was obtained from embryos cultured for 48 h before the transfer. The results indicate that the viability of in vitro-produced porcine embryos is decreased by IVC after IVF; however, these embryos have competence to develop to term. An improved IVC system of porcine IVM/IVF oocytes is needed to generate advances in this field.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In vitro porcine embryo production systems that generate embryos as viable as in vivo embryos will contribute to research in reproductive physiology, agriculture, and biotechnologies, including transgenesis and cloning. Developmental competence or viability of in vitro-matured (IVM) and in vitro-fertilized (IVF) porcine oocytes was confirmed and first reported by Mattioli et al. [1]. The birth of piglets has been accomplished from IVM/IVF embryos cultured in vitro (IVC) to the 2- to 4-cell stage [2]. Some laboratories [2–4] have succeeded in producing piglets from cleaved embryos that were transferred after IVM/IVF and IVC for 24–36 h. Transfer of 8-cell- to morula-stage embryos after 96 h culture has resulted in living offspring [5]. These successes in reproduction are attributable to recent advances in IVM and IVC systems. IVM systems for porcine follicular oocytes have been improved in recent years, emphasizing the importance of the porcine follicular fluid fraction and cysteine in the maturation medium [2] and synchronization of early meiosis in oocytes [4]. On the other hand, North Carolina State University (NCSU) solutions containing taurine, hypotaurine, or sorbitol for porcine embryo cultures have also been developed using in vivo-derived embryos [6–9]. These innovations were introduced to in vitro production systems (IVC) of porcine embryos after IVM and IVF. Although in vitro production systems have been improved, there is still the problem of low embryo survival. In addition, no one has succeeded in generating piglets after transfer of in vitro-produced blastocysts. In this study, we examined the ability of IVM/IVF embryos cultured for 0, 24, or 48 h to develop after embryo transfer to recipients.

	MATERIALS AND METHODS

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Oocyte Collection and IVM

Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 35°C. Cumulus-oocyte complexes (COCs) were collected from follicles 3–5 mm in diameter in Medium 199 (with Hanks' salts; Gibco, Life Technologies Inc., Grand Island, NY) supplemented with 10% (v:v) fetal bovine serum (Gibco), 20 mM Hepes (Dojindo Laboratories, Kumamoto, Japan), 100 IU/ml penicillin G potassium (Sigma Chemical Co., St. Louis, MO), and 0.1 mg/ml streptomycin sulfate (Sigma) [10, 11]. IVM was carried out basically by the method of Funahashi et al. [4]. Collected COCs were cultured in modified NCSU-37 solution [9] containing 10% porcine follicular fluid, 0.6 mM cysteine, 1 mM dibutyryl cAMP (Sigma), 10 IU/ml eCG (PMS 1000 IU; Nihon Zenyaku Kogyo, Koriyama, Japan), 10 IU/ml hCG (Puberogen 500 U; Sankyo, Tokyo, Japan), and antibiotics for 20–22 h. They were subsequently cultured in modified NCSU-37 solution without dibutyryl cAMP and hormones for 24 h. The culture was carried out at 39°C under 5% CO₂ in air.

IVF with Frozen-Thawed Epididymal Spermatozoa

Boar epididymides from the Meishan breed were collected at a slaughterhouse. They were transferred to the laboratory at 25°C within 1 h. Epididymal spermatozoa were collected and frozen as described previously [12]. Semen was extruded from the distal portion of the cauda epididymidis and diluted with Niwa and Sasaki Freezing-II extender containing 18.5% (v:v) egg yolk, 0.75% (v:v) Equex Stem (Noba Chemical Sales Inc., Scituate, ME), and 3.0% (v:v) glycerol. Sperm suspensions were loaded into 0.25-ml plastic straws (IMV, L'aigle, Cedex, France) after adjustment of the sperm concentration to 1 x 10⁹/ml and placed in liquid nitrogen vapor for 10 min; they were then plunged into liquid nitrogen. IVF was carried out as described previously [10]. After thawing at 37°C, spermatozoa were preincubated for 1 h in Medium 199 that had been adjusted to pH 7.8 [13]. A portion (10 µl) of preincubated sperm was introduced into the fertilization medium, 90 µl Bracket and Oliphant solution [14] supplemented with 10 mg/ml BSA (fraction V; Sigma) containing 10 IVM oocytes surrounded by expanded cumulus cells. The final sperm concentration was 1 x 10⁶/ml. Insemination was carried out for 5 h. To examine the results of the IVF, some inseminated oocytes were transferred to IVC medium, NCSU-37 solution containing 4 mg/ml BSA and 50 µM ß-mercaptoethanol (Sigma) and subsequently cultured at 38.5°C under conditions of CO₂ and N₂ adjusted to 5% and 90%, respectively, for 5 h [15]. Then they were fixed with acetic alcohol (1:3), stained with 1% aceto-orcein, and examined under a phase-contrast microscope.

Embryo Culture

To examine the ability of the IVM/IVF embryos to develop in vitro, some embryos were subsequently cultured in vitro for 24, 48, and 144 h (Days 1, 2, and 6, respectively). After the culture, they were fixed, stained, and examined for embryonic stage and total cell number. An embryo consisting of cells in which cytoplasm was stainable with orcein and showed compaction was defined as a morula; an embryo having a clear blastocoele was defined as a blastocyst.

Synchronization of Recipients and Embryo Transfer

Early-pregnant crossbred gilts were used as embryo recipients. Estrus synchronization was carried out by an i.m. injection of 0.18 mg prostaglandin F₂ analogue (Cloprostenol; Planate, Schering-Plough Corporation, Kenilworth, NJ) on the 16th to 45th day of gestation, followed by a second injection of 0.09 mg Cloprostenol 24 h later [16,17]. One thousand IU of eCG (PMS 1000 Tani NZ, Nihon Zenyaku, Japan) was administered i.m. at the same time as the second Cloprostenol injection. Ovulation was induced by i.m. injection of 500 IU hCG (Puberogen) 72 h after eCG injection. Ovulation was expected on the day of IVF (Day 0) at 40–44 h after the hCG injection. Day 0 embryos without culture or Day 1 and 2 (cultured for 24 and 48 h, respectively) embryos after IVC were transferred to transfer medium, NCSU-37 solution supplemented with 4 mg/ml BSA and 25 mM Hepes, adjusted to 280 osmol/kg. They were transported to the farm within 2 h by car. After confirmation of immobility of spermatozoa, which were attaching to the zona pellucida, 100 Day 0 and Day 1 embryos without selection were surgically introduced per recipient into oviducts 45 and 69 h after hCG injection, respectively. In addition, cleaved Day 2 embryos obtained from 100 IVM/IVF oocytes were surgically transferred per recipient into oviducts 93 h after hCG injection.

Examination of Embryo Survival

On the 29th day (Day 29) after IVF, the uteri of some recipients were surgically examined; pregnant females were then subjected to hysterectomies in order that fetuses could be counted and weighed. Pregnancy of the other recipients was diagnosed using an ultrasound pregnancy detector (Medata Systems Ltd., Arundel, West Sussex, UK), and the pregnant females were allowed to go to term.

Statistical Analysis

All data were subjected to ANOVA using General Linear Models procedures of the Statistical Analysis System. Weights of Day 29 fetuses and newborn piglets were examined by the least significant difference test. Developmental rates calculated from the number of fetuses or piglets in relation to the total number of inseminated oocytes were analyzed by the Duncan's Multiple Range test after transformation using arcsin of percentage [18].

	RESULTS

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Oocyte Maturation, Fertilization, and Embryo Culture

As shown in Table 1, about 70% of examined oocytes were identified as matured oocytes. The percentage of penetration in relation to the total number of examined oocytes was 30%. The percentages of monospermy and male pronuclear formation were 18% and 28%, respectively. All of the monospermic oocytes formed a well-developed male pronucleus. The percentage of oocytes fertilized normally (oocytes with monospermic penetration having male and female pronucleus and 2 polar bodies) in relation to number of inseminated oocytes was determined to be 18% at 10 h postinsemination. As Table 2 shows, 2- to 4-cell-stage embryos were observed (5%) on Day 1 post-IVF and became dominant (31%) at Day 2. Degenerated or fragmented oocytes appeared from Day 1 (18%), and the incidence increased to 85% of inseminated oocytes until Day 6 (Table 2). On Day 6, the percentage of embryos that had developed to the morula or blastocyst stage was 14% of inseminated oocytes. Total cell numbers of blastocysts developed in vitro ranged from 9 to 114, and the average was 39.4. The other embryos were degenerated or fragmented after unequal cleavage of cytoplasm and showed no stainability with orcein.

Effect of Culture Period on Embryo Survival

As shown in Table 3, during early gestation until Day 29, the incidence of pregnancy (43–71%) did not differ between experimental groups; however, the developmental rate to fetuses for oocytes inseminated and transferred without any culture was significantly higher (6.7%, p < 0.05) than for those transferred after culture for 24 and 48 h (1.7% and 2.0%, respectively). The fetal weights did not differ (1.0–1.2 g) among the experimental groups. As Table 4 shows, 1 of 2 recipients was found to be pregnant after the transfer of Day 2 embryos, whereas both recipients were pregnant in the other two groups (Day 0 and Day 1 embryos). No farrows were obtained in the Day 2 group, whereas 2 and 1 recipients farrowed in the Day 0 and Day 1 groups, respectively. The weight of newborn piglets did not differ between the Day 0 and Day 1 groups. Developmental rate to term was 4.0–4.5%.

	DISCUSSION

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The percentage of penetration was lower (30%, Table 1) than that in our previous study (70%) [10] using the same IVF system except for the use of different batches of epididymal spermatozoa. The incidence of monospermy (18%) was, however, the same as in the previous study (15%). Since a higher ability of sperm to penetrate causes a higher percentage of polyspermic fertilization [19], selecting sperm batches carefully is necessary for studies using IVM/IVF embryos. Normal IVF oocytes (oocytes having both a single male and female pronucleus and 2 polar bodies) were obtained with a frequency of 18% of inseminated oocytes. In the present study, inseminated oocytes developed to blastocysts after IVC for 144 h in the proportion (13%, Table 2) expected. However, embryo quality should be discussed, rather than the quantity of embryo production, because the total cell number after culture was small (39 after culture for 144 h), resulting in poor viability of the fetuses (6.7%, Table 3) even after embryo transfer on Day 0.

After insemination for 5 h and transportation to the farm for 2 h before embryo transfer, we confirmed the immobility of spermatozoa, which were attaching to the zona pellucida of Day 0 zygotes. At that time (7 h postinsemination), spermatozoa attached or not attached to the zona pellucida had lost their motility. In the batch of spermatozoa we used, spermatozoa lost their motility rapidly during the 2 h after thawing (unpublished results) but during this time could fertilize oocytes [19]. It can be concluded that there is no possibility of fertilization of ovulated oocytes by spermatozoa detached from transferred Day 0 zygotes.

Fragmentation of oocytes was considered to result from abnormal activation of oocytes by sperm penetration or after parthenogenesis [20], and subsequent death. It is very difficult to distinguish healthy embryos beyond the 2- or 4-cell stage from fragmented oocytes under stereomicroscopic observation. Therefore, we transferred Day 2 embryos as cleaved oocytes (Tables 3 and 4). Among the cleaved oocytes transferred to recipients, there were, most likely, both living embryos beyond the 2-cell stage and some fragmented oocytes as well. The percentage of blastocyst formation on Day 6 was low (13%) compared to that reported by Funahashi and Day (34%) [21]; however, the quality of embryos (measured as total cell number of blastocysts) was 39 and seems to be equal to that in other studies (total cell numbers were 30 [22] and 37 [23]). These results suggest that selection of the sperm batch for IVF resulting in a higher incidence of monospermic penetration is important for higher incidences of blastocyst formation but not for quality of in vitro-produced embryos. The total number of cells of in vitro-produced embryos has been shown to be reduced compared to that for their in vivo counterparts [24]. When 2- to 4-cell-stage embryos after IVM/IVF/IVC were transferred and recovered after 4 days, more than 48% of transferred embryos had developed to morulae or blastocysts [1]. These observations suggest that the culture system rather than the IVF procedure seems to be causing differences in cell number of cultured embryos [24]; however, the effect of in vivo conditions during early cleavages remains unclear. Examination of the mechanisms of supporting embryonic development in oviductal circumstances may give a hint for the progress of IVC systems.

In gilts, most prenatal losses occur by Day 30 postmating, which is comparable to Day 29 in the present study [25]. Therefore, we assessed the developmental ability of IVM/IVF(/IVC) embryos on Day 29. The developmental rate of fetuses at Day 29 from IVM/IVF embryos transferred without culture (6.7%) was about 3.6 times higher than those of embryos cultured for 24 or 48 h (1.7% and 2.0%, respectively) (Table 3). When normality of development to fetuses was analyzed by fetal weights, there was no significant difference among the experimental groups. The results indicate that the viability of porcine embryos produced in vitro is decreased by IVC after IVF; however, embryos that survive after IVC could develop to normal fetuses during early gestation. These results suggest that there are one or more detrimental factors in the IVC system after IVM/IVF and that the IVC system used in this study is not the optimal one. In other porcine embryo culture systems, the viabilities of cultured blastocysts were lower than those of noncultured embryos [26]. In addition, some fetuses were subjected to histological examination (data was not shown), and organs were developing well; however, there seemed to be some delay in comparison to development of in vivo-derived fetuses at the same gestation [27], because gonads had not developed to testes or ovaries.

Developmental competence of IVM/IVF/IVC porcine embryos has been shown in previous studies, and piglets have been obtained. Investigators cultured IVM/IVF oocytes for 36–48 h [1–4] or 96 h [5] and then transferred cleaved embryos (2- to 4-cell-stage embryos or 8-cell- to morula-stage embryos, respectively) to recipients. The rate of embryo development to piglets was estimated as 0.3–1.1% [1–3], which was calculated from the number of piglets in relation to inseminated oocytes. In the present study, no piglets were born when embryos were cultured for 48 h and transferred; however, higher percentages of developmental ability were obtained by transferring Day 0 and Day 1 embryos (4.5% and 4.0%, respectively). There was no significant difference in developmental rates between Day 0 and Day 1 embryos because replications of experiments in embryo transfer were too small in the present study (Table 4). The weight of newborn piglets was not different between those derived from Day 0 and Day 1 embryos. These results of the present study indicate that embryo development to term has a tendency to decrease with increasing period of IVC; on the other hand, newborn piglets were not different in weight when embryos produced in vitro were transferred on Day 0 and Day 1.

In conclusion, the viability of porcine in vitro-produced (IVM/IVF/IVC) embryos is decreased by IVC after IVF; however, these embryos have competence to develop to term. An improved IVC system for porcine IVM/IVF oocytes is needed to generate advances in this field.

	ACKNOWLEDGMENTS

 
The authors would like to thank Dr. A. Onishi for providing us with Meishan epididymides, Dr. D.A. Vaughan for help with English, Ms. T. Aoki for technical assistance, and Mr. K. Takagi at Sunrise Farm for preparing recipients.

	FOOTNOTES

 
¹ Correspondence: Kazuhiro Kikuchi, Department of Genetic Resources II, National Institute of Agrobiological Resources, Kannondai 2–1-2, Tsukuba, Ibaraki 305–8602, Japan. FAX: 81 298 38 7408; kiku{at}abr.affrc.go.jp

Accepted: September 15, 1998.

Received: June 26, 1998.

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